JP2012510291A - Novel Lactobacillus plantarum and composition containing the same - Google Patents

Novel Lactobacillus plantarum and composition containing the same Download PDF

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JP2012510291A
JP2012510291A JP2011539440A JP2011539440A JP2012510291A JP 2012510291 A JP2012510291 A JP 2012510291A JP 2011539440 A JP2011539440 A JP 2011539440A JP 2011539440 A JP2011539440 A JP 2011539440A JP 2012510291 A JP2012510291 A JP 2012510291A
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lactobacillus plantarum
lactic acid
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ポン−ジュン キム
ホン ウン チョン
サン−ヒョン セオ
カン−ピョ イ
クァン−ウ ファン
テ−ヨン ウォン
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Abstract

【解決手段】本発明は、ラクトバシルロス・プランタラムCJLP56(Lactobacillus plant arumCJLP56) KCTC 11402BP、前記乳酸菌を含む腸疾患治療用組成物、および前記乳酸菌を含む免疫増強用組成物を提供する。
【選択図】図1
The present invention provides a Lactobacillus plant arum CJLP56 (CJLP56) KCTC 11402BP, a composition for treating intestinal diseases containing the lactic acid bacterium, and an immune enhancing composition containing the lactic acid bacterium.
[Selection] Figure 1

Description

本発明は、新規なラクトバチルス・プランタラム及びこれを含む組成物に係り、より具体的には腸疾患及び免疫疾患の予防治療に有用な新規なラクトバチルス・プランタラム及びこれを含む組成物関することだ。   The present invention relates to a novel Lactobacillus plantarum and a composition containing the same, and more particularly to a novel Lactobacillus plantarum useful for the prevention and treatment of intestinal diseases and immune diseases and a composition containing the same. Thing.

キムチのような伝統発酵食品に豊富に存在する乳酸菌は、人体の消化界に共生しながら繊維質および複合蛋白質らを分解して重要な栄養成分で作る役割を担当して、このように人を含んだ動物の胃腸管内で宿主の腸内微生物環境を改善して宿主の健康に有益な影響を与え生きている微生物を通称してプロバイオティクス(probiotics)とする。プロバイオティクス(probiotics)とて効果があるためには経口で摂取して小腸に到達して腸表面に付着して維持されなければならないので、基本的に耐酸性、耐胆汁酸性、および腸皮細胞付着能力が優秀でなければならない。   Lactic acid bacteria, which are abundant in traditional fermented foods such as kimchi, are in charge of the role of breaking down fiber and complex proteins and making them with important nutritional components while coexisting with the digestive world of the human body. Probiotics are commonly referred to as living microorganisms that have beneficial effects on the health of the host by improving the intestinal microbial environment of the host in the gastrointestinal tract of the contained animal. In order to be effective as probiotics, it must be taken orally, reach the small intestine and remain attached to the intestinal surface, so it is basically acid resistant, bile acid resistant, and intestinal skin Cell attachment ability must be excellent.

キムチのような伝統醗酵食品で発見される代表的なプロバイオティクスとてラクトバチルス属(Lactobacillus sp.)乳酸菌がある。ラクトバチルス属微生物は、同型または異形発効をする乳酸桿菌として人を含んだ動物の腸管および乳製品や野菜の発効過程でよく見ることができる。ラクトバチルス属微生物は、腸内pHを酸性で維持させて大腸菌(E.coli)やクロストリジュウム(Clostridium)と同じ有害菌の繁殖を抑制して下痢と便秘を改善するだけでなくビタミン合成、坑癌作用、血清コレステロール低下などの役割をすると知られている。乳酸桿菌によって生産されるアシドリン(acidophillin)は、疫痢菌、サルモネラ菌、葡萄状球菌、大腸菌などの成長を阻害すると知られている。また、下痢原因菌の増殖を抑制して腸内菌総を正常化することで下痢を立ち止らせる作用をする(非特許文献1及び非特許文献2).
ラクトバチルス属微生物の前記特性を利用して生菌剤および家畜飼料で開発しようと思う研究が活発に進行している。家畜の細菌性下痢病は、増体率減少と斃死を誘発する。したがって、これを予防して家畜の生産を高めようと飼料に抗生剤を添加することが一般的に広く行われてきた。しかし、抗生物質に対する耐性菌の出現と畜産物内の残留抗生物質などの問題のために飼料内抗生物質の使用を規制して有機的な家畜飼養法が強調されている傾向だ(特許文献1)(非特許文献3)。
Lactobacillus sp. Lactic acid bacteria are typical probiotics found in traditional fermented foods such as kimchi. Lactobacillus microorganisms can often be seen in the intestinal tract of animals including humans as the lactobacilli that have the same or different forms of action, and in the process of action of dairy products and vegetables. Lactobacillus microorganisms not only improve the diarrhea and constipation by maintaining the intestinal pH acidic and suppressing the growth of the same harmful bacteria as E. coli and Clostridium. It is known to play a role such as cancer action and serum cholesterol lowering. Acidophillin produced by Lactobacillus is known to inhibit the growth of Shigella, Salmonella, Staphylococcus and Escherichia coli. Moreover, it acts to stop diarrhea by suppressing the growth of diarrhea-causing bacteria and normalizing the intestinal bacteria total (Non-Patent Document 1 and Non-Patent Document 2).
Research that intends to develop live fungi and livestock feed using the aforementioned characteristics of Lactobacillus microorganisms is actively underway. Bacterial diarrhea in livestock induces decreased body weight gain and moribundity. Therefore, it has been widely practiced to add antibiotics to feed to prevent this and increase livestock production. However, due to the emergence of antibiotic-resistant bacteria and problems such as residual antibiotics in livestock products, organic livestock feeding methods tend to be emphasized by regulating the use of antibiotics in feed (Patent Document 1). (Non-Patent Document 3).

また、ラクトバチルス属微生物と同じ乳酸菌は、免疫増強効果を持つことでも知られている。最近全世界的に環境汚染とインスタントフード摂取の増加などの影響で予測される免疫調節以上と関連したアレルギーおよびアトピー疾患が急激に増加していて、韓国でも同じようにこのような疾患が増加傾向にある。最近ヨーロッパでは乳酸菌を經口投与して病気を治療する微生物治療(bacteriotheraphy)の一環で乳酸菌で病の症状を緩和したり改善する努力が成り立っている。ラクトバチルス・ラムノサスGG(Lactobacillus rhamnosus GG)を乳児に投与時アトピー発生が半分水準で減少したし(非特許文献4)、すでにアトピー湿疹が進行中の子供にラクトバチルス・ラムノサス(Lactobacillus rhamnosus)とラクトバチルス・ロイテリ(L. reuteri)を投与する場合、湿疹部位および程度が減少するという報告がある(非特許文献5)。   The same lactic acid bacteria as Lactobacillus microorganisms are also known to have an immune enhancing effect. Allergies and atopy diseases related to more than the immunomodulation predicted by the influence of environmental pollution and increase of instant food intake are increasing rapidly worldwide, and such diseases are also increasing in Korea as well. It is in. Recently, in Europe, efforts have been made to alleviate or improve disease symptoms with lactic acid bacteria as part of microbial treatment (bacteriotheraphy), which treats illnesses by mouth. When Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) was administered to infants, the incidence of atopy was reduced to a half level (Non-patent Document 4), and Lactobacillus rhamnosus and Lactobacillus rhamnosus and Lactobacillus rhamnosus When administering Bacillus reuteri (L. reuteri), there is a report that the site and degree of eczema are reduced (Non-patent Document 5).

このような乳酸菌の免疫増強効果に対するメカニズム(mechanism)に対してずっと研究が進行されていて、具体的なメカニズムに対してはまだ明確に明らかになることはなかったが、おおむね經口的に流入して腸内で棲息することで腸管免疫系に影響を及ぼすと知られている。例をあげれば、ヨーグルトを通した乳酸菌摂取はPeyer's patchのリンパ球らの坑菌活性を増加させると知られていて、実験動物および人を対象に実行された一部研究らによれば乳酸菌は、IgAの反応を強化させると知られている。また、乳酸菌は、先天免疫および適応免疫皆に影響を及ぼす。腸管免疫系の先天免疫反応(innate immunity)では病原菌を貪食して死滅させることで感染に対抗して健康を維持する役割をすると知られている。適応免疫(adaptive immunity)では抗原を分解してTリンパ球に提示する役割をするマクロファージを活性化して多様なサイトキン(cytokine)、特にインタールーキンIL-12、IL-18生産を増加させるが、これは乳酸菌細胞壁構成成分中一部がマクロファージでNF-κB、STAT信号傳達を活性化させることによってサイトキン(cytokine)の生成が増加すると知られている。また、乳酸菌は、専門的な抗原提示細胞としてリンパ節(lymph node)および消化器系の粘膜にたくさん存在する樹状細胞(dendritic cell)でIL-12、IL-18、TNF-α生成を増加させるのはもちろんMHC class IIおよびB7-2とともにTリンパ球(lymphocyte)を活性化させる表面分子の発現も増加させると知られている(非特許文献6)。   Research on the mechanism for the immune enhancement effect of lactic acid bacteria has been ongoing, and the specific mechanism has yet to be clearly clarified. Thus, it is known that inhabiting the intestine affects the intestinal tract immune system. For example, lactic acid bacteria intake through yogurt is known to increase antibacterial activity of lymphocytes of Peyer's patch. According to some studies conducted on laboratory animals and humans, lactic acid bacteria are , Known to enhance the reaction of IgA. Lactic acid bacteria also affect both innate and adaptive immunity. Innate immunity of the intestinal tract immune system is known to play a role in maintaining health against infection by phagocytosing and killing pathogenic bacteria. Adaptive immunity activates macrophages, which are responsible for degrading antigens and presenting them to T lymphocytes, increasing the production of various cytokines, especially interleukins IL-12 and IL-18. This is known to increase the production of cytokines by activating NF-κB and STAT signals in macrophages, which are part of lactic acid bacteria cell wall constituents. Lactic acid bacteria also increase IL-12, IL-18, and TNF-α production in the lymph nodes and dendritic cells that are present in the mucosa of the digestive system as specialized antigen-presenting cells. It is known that, together with MHC class II and B7-2, expression of surface molecules that activate T lymphocytes is also increased (Non-patent Document 6).

Tリンパ球は、適応免疫(adaptive immunity)の中心になる細胞として、適応免疫は、細胞性免疫のTh1反応と抗体性免疫のTh2反応で分けることができる。Th1およびTh2それぞれの反応で抗原提示細胞(Antigen Presenting Cell)が生産するサイトウカインが互いに違って、Th1反応では、IL-12、IL-18、インターフェロン(IFN)生産が優勢でTh2反応では、PGE2、IL-4、IL-10生産が優勢だ。このようなTh1反応およびTh2反応は、適切な均衡が成り立つべきで、均衡がこわれる場合、各種免疫疾患が現れると知られている。Th1細胞は、主に感染症と戦う反面Th2細胞は、主にアレルギーと炎症性反応に関与する。これらが正常に作用する時にはTh2細胞は、ホコリおよびその他願わない物質から身体を保護するが、万一、これら細胞が過度な活性を現わす場合IgE抗体生産が増加して人体に威嚇にならなかった蛋白質(例:花粉、食物など)にアレルギー反応を誘発させる。したがって、Th1反応とTh2反応は、必ず均衡を維持するべきで、万一、この中で一つが過剰や他の一つが不足すれば病気が誘発される。また、続くストレスによってコルチゾールが持続的に遊離すればTh1反応が低下してTh2反応が増加することになって、癌、アトピー、アレルギーおよび自家免疫疾患が誘発される(非特許文献7)。   T lymphocytes are the cells that play a central role in adaptive immunity, and adaptive immunity can be divided into Th1 responses of cellular immunity and Th2 responses of antibody immunity. Cytokines produced by antigen-presenting cells in Th1 and Th2 reactions differ from each other.In the Th1 reaction, IL-12, IL-18, and interferon (IFN) production predominate, and in the Th2 reaction, PGE2 IL-4 and IL-10 production is dominant. It is known that an appropriate balance should be established between the Th1 response and the Th2 response, and various immune diseases appear when the balance is broken. Th1 cells primarily fight infection, whereas Th2 cells are primarily involved in allergies and inflammatory reactions. When these function normally, Th2 cells protect the body from dust and other unwanted substances, but in the unlikely event that these cells exhibit excessive activity, IgE antibody production increases and does not threaten the human body. Causes allergic reactions to proteins (eg, pollen, food, etc.). Therefore, the Th1 and Th2 responses should be kept in balance, and if one of them is excessive or the other is insufficient, the disease is induced. Further, if cortisol is continuously released by the subsequent stress, the Th1 response decreases and the Th2 response increases, and cancer, atopy, allergies and autoimmune diseases are induced (Non-patent Document 7).

In vivo実験によれば乳酸菌は、Tリンパ球でTh1サイトキン(cytokine)のIFN-γの生成を増加させて、Th2サイトキンのIL-4、IL-5生成を抑制するという(非特許文献8)。また、他の実験でもTh2反応動物モデルの卵アルブミン(Ovalbumin)を投与してTh2反応で偏向したマウス(ovalbumin-primed mice)に乳酸菌を経口投与時脾臟細胞(splenocyte)でIFN-γが増加したし、IL-4、IL-5およびIgEが減少したし卵アルブミン(Ovalbumin)を投与してTh2反応で偏向したマウスで摘出した脾臟細胞を乳酸菌と共に培養時にもサイトキンおよびIgEの変化が経口投与実験と同一だと知られている。しかし、Tリンパ球だけを乳酸菌と培養時IFN-γ生成が有意性あるように増加しなかったのでTリンパ球のIFN-γ生成には、マクロファージ、樹状細胞のような抗原提示細胞が必ず必要なことで見なされる(非特許文献9)。一方IL-12およびIL-18は、Th0リンパ球をTh1リンパ球で分化させるのに重要なサイトキンとしてマクロファージまたは受賞細胞で生成されて、脾臟細胞またはマクロファージ培養時乳酸菌を処理すれば濃度依存的にIL-12、IL-18およびIFN-αなどの生成が増加すると知られている。このように乳酸菌は、マクロファージでIL-12、IL-18およびIFN-αなどの生成を増加させるのでTh1細胞での分化を促進してこれらのIFN-γ生成を誘導するので、Th2が優勢な状況でTh1/Th2均衡を合わせる作用をする(非特許文献6)。したがって、乳酸菌は、Th2反応過剰によるTh1/Th2不均衡に誘発される癌、アトピー、アレルギー、および自家免疫疾患を予防または治療するのに役に立つと知られている。   According to in vivo experiments, lactic acid bacteria increase IFN-γ production of Th1 cytokines in T lymphocytes and suppress IL-4 and IL-5 production of Th2 cytokins (non-patent literature). 8). In other experiments, IFN-γ was increased in splenocytes when orally administered lactic acid bacteria to mice that were administered ovalbumin (Ovalbumin), a Th2-reactive animal model, and orally administered ovalbumin-primed mice. In addition, spleen splenocytes isolated in mice that had decreased IL-4, IL-5, and IgE, and were ovalbumin-administered and biased by the Th2 reaction, were orally administered with cytokines and IgE when cultured with lactic acid bacteria. It is known to be identical to the experiment. However, only T lymphocytes did not increase so that IFN-γ production was significant when cultured with lactic acid bacteria, so antigen-presenting cells such as macrophages and dendritic cells must be used for IFN-γ production of T lymphocytes. It is regarded as necessary (Non-Patent Document 9). IL-12 and IL-18, on the other hand, are produced in macrophages or award-winning cells as important cytokines for differentiating Th0 lymphocytes with Th1 lymphocytes, and are concentration-dependent if treated with spleen sputum cells or lactic acid bacteria during macrophage culture. It is known that the production of IL-12, IL-18 and IFN-α is increased. Thus, lactic acid bacteria increase the production of IL-12, IL-18, IFN-α, etc. in macrophages, thus promoting differentiation in Th1 cells and inducing these IFN-γ production, so Th2 is dominant. It acts to match the Th1 / Th2 balance in the situation (Non-Patent Document 6). Thus, lactic acid bacteria are known to be useful in preventing or treating cancer, atopy, allergies, and autoimmune diseases induced by Th1 / Th2 imbalance due to excessive Th2 response.

韓国特許公開1998-78358Korean Patent Publication 1998-78358

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これに、本発明者などは、従来公示されている乳酸菌に比べてTh2反応過剰によるTh1/Th2不均衡を調節する効果が非常に優秀な新しい乳酸菌を開発するために研究した結果、伝統発酵食品から新規なラクトバチルス属酸菌菌株を分離、同定して本発明を完成することになった。   In addition, the present inventors have conducted research to develop a new lactic acid bacterium that is very effective in regulating Th1 / Th2 imbalance caused by excessive Th2 reaction compared to the lactic acid bacterium that has been publicly announced. The present invention was completed by isolating and identifying a novel Lactobacillus acidobacillus strain.

したがって、本発明の目的は、プロバイオティクスとしての基本性質の耐酸性、耐胆汁酸性、腸上皮細胞付着能が優秀なだけでなく、免疫増強効果、特にTh2反応過剰によるTh1/Th2不均衡を調節する効果が優秀な新規なラクトバチルス属乳酸桿菌菌株を提供することである。   Therefore, the object of the present invention is not only excellent in acid resistance, bile acidity and intestinal epithelial cell adhesion ability, which are basic properties as probiotics, but also enhances immune enhancement effects, particularly Th1 / Th2 imbalance due to excessive Th2 reaction. It is to provide a novel Lactobacillus genus Lactobacillus having an excellent effect of regulating.

本発明の他の目的は、前記ラクトバチルス属乳酸桿菌菌株を含む腸疾患の予防または治療用組成物を提供することである。   Another object of the present invention is to provide a composition for preventing or treating intestinal diseases comprising the Lactobacillus strain of Lactobacillus.

本発明のまた他の目的は、前記ラクトバチルス属乳酸桿菌菌株を含む免疫増強用組成物を提供することである。   Another object of the present invention is to provide a composition for enhancing immunity comprising the Lactobacillus strain of Lactobacillus.

前記目的を達成するために、本発明は、ラクトバチルス・プランタラムCJLP 56(Lactobacillus plantarumCJLP56)(寄託機関:生命工学研究院遺伝子銀行、寄託日時:2008.10.16、受託番号:KCTC 11402BP)を提供する。   In order to achieve the above object, the present invention provides Lactobacillus plantarum CJLP 56 (Deposit organization: Biobank of Biotechnology, Deposit date: 2008.10.16, Deposit number: KCTC 11402BP). .

また、本発明は、前記ラクトバチルス・プランタラムCJLP56を含む腸疾患の予防または治療用組成物を提供する。   The present invention also provides a composition for preventing or treating intestinal diseases comprising the Lactobacillus plantarum CJLP56.

また、本発明は、ラクトバチルス・プランタラムCJLP56を含む免疫増強用組成物を提供する。   The present invention also provides an immunopotentiating composition comprising Lactobacillus plantarum CJLP56.

以下、本発明をより詳細に説明する。   Hereinafter, the present invention will be described in more detail.

本発明にともなうラクトバチルス・プランタラムCJLP56(Lactobacillus plantarumCJLP56)は、伝統発酵食品から分離および同定されたラクトバチルス・プランタラムの新規な菌株であることを特徴とする。前記伝統発酵食品では、キムチ、野菜発効物、味噌、醤油、清麹醤または塩辛などがあるが、これに限定されるのではない。   Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) according to the present invention is a novel strain of Lactobacillus plantarum isolated and identified from traditional fermented foods. Examples of the traditional fermented food include kimchi, vegetable effect products, miso, soy sauce, neat soy sauce, and salted salt, but are not limited thereto.

本発明のラクトバチルス・プランタラムCJLP56は、微生物の同情および分類のための16S rRNA塩基配列分析結果ラクトバチルス・プランタラム標準菌株(Lactobacillus plantarum NBRC15891T、GenBank accession number AB326351)と最も高い相同性(99.9%)を現わしてラクトバチルス・プランタラムと最も高い分子系統学的柔軟関係を見せた。したがって、前記微生物をラクトバチルス・プランタラム(Lactobacillus plantarum)で同定して、ラクトバチルス・プランタラムCJLP56で命名したし、生命工学研究院遺伝子銀行に2008年10月16日付で寄託した(受託番号KCTC 11402BP)。ラクトバチルス・プランタラムCJLP56の16S rRNA遺伝子の塩基配列は、本明細書に添付された塩基配列目録SEQ ID NO.1のようだ。   Lactobacillus plantarum CJLP56 of the present invention is the highest homology (99.9%) with the 16S rRNA nucleotide sequence analysis results Lactobacillus plantarum NBRC15891T, GenBank accession number AB326351) for sympathy and classification of microorganisms ) Showed the highest molecular phylogenetic flexibility with Lactobacillus plantarum. Therefore, the microorganism was identified by Lactobacillus plantarum, named by Lactobacillus plantarum CJLP56, and deposited at the Biotechnology Research Institute Genetic Bank on October 16, 2008 (Accession No.KCTC). 11402BP). The base sequence of the 16S rRNA gene of Lactobacillus plantarum CJLP56 seems to be the base sequence listing SEQ ID NO. 1 attached hereto.

本発明のラクトバチルス・プランタラムCJLP56は、クラミ陽性菌であり好気的条件と嫌気的条件で全部成長が可能な通性嫌気性(facultive anaerobe)であり胞子を形成しないで運動性がなくて細胞の形態は、桿菌である。   Lactobacillus plantarum CJLP56 of the present invention is a cultivar-positive bacterium that is capable of growing under aerobic and anaerobic conditions, is facultive anaerobe, has no motility without forming spores The cell morphology is Neisseria gonorrhoeae.

ラクトバチルス・プランタラムCJLP56のより具体的な形態および生理学的特性は、当該技術分野の通常の方法により分析した結果表1と同じであることが分かった。   The more specific form and physiological characteristics of Lactobacillus plantarum CJLP56 were found to be the same as Table 1 as a result of analysis by ordinary methods in the art.

Figure 2012510291
Figure 2012510291

本発明のラクトバシルロス・プランタラム CJLP56は、長期間安定的に保存するためには水にグリセロール成分を一定量混合して作った保管溶液に菌体を解いて-70℃で保管したり滅菌された10%脱脂乳に懸濁して凍結乾燥することが好ましい。   Lactobacillus plantarum CJLP56 of the present invention can be stored at -70 ° C by dissolving the cells in a storage solution prepared by mixing a certain amount of glycerol component with water in order to stably store for a long period of time. It is preferable to suspend and freeze-dry in 10% skimmed milk.

また、本発明のラクトバシルロス・プランタラム CJLP56は、プロバイオティクスとして乳酸菌の一般的な整腸効果および免疫増強効果を持つ。   Further, Lactobacillus plantarum CJLP56 of the present invention has a general intestinal effect and immunity enhancing effect of lactic acid bacteria as probiotics.

本発明において、'プロバイオティクス(probiotics)'は、人を含んだ動物の胃腸管内で宿主の腸内微生物環境を改善して宿主の健康に有益な影響を与え生きている微生物という意味で理解される。プロバイオティクスは、プロバイオティク活性を持って生きている微生物で単一または複合菌株形態で人や動物に乾燥された細胞形態や発効産物形態で給与される場合、宿主の腸内菌総に有益な影響を及ぼすことができる。このようなプロバイオティク微生物であるためには一番目で、胃液と胆汁での影響を少なく受けながら胃を通過して腸で生存できるかを確認して、腸内に定着して生存が可能なのかを確認して宿主の腸内菌総に有益な影響を及ぼすべきだ。したがって、胃酸に対する耐酸性、胆汁酸に対する耐胆汁酸性および腸上皮細胞に対する付着性を持たなければならない。二番目で、プロバイオティク微生物であるためには、微生物の安全性に問題があってはならなくて、これと関連しては一般的にゼラチン液化反応検査、フェニルアラニン脱アミン生成検査、アンモニア生成検査、溶血性実験検査などが遂行される。本発明にともなうラクトバシルロス・プランタラムCJLP56は、優秀な耐酸性、胆汁酸に対する耐胆汁酸性腸上皮細胞に対する付着性を持つだけでなく、ゼラチン液化反応検査、フェニルアラニン脱アミン生成検査およびアンモニア生成検査で陰性で現れたし、溶血性実験検査では病原性と関係がないと判定されるα-溶血で確認されて安全なことが分かった。   In the context of the present invention, 'probiotics' is understood to mean living microorganisms that improve the intestinal microbial environment of the host in the gastrointestinal tract of animals, including humans, and have a beneficial effect on the health of the host. Is done. Probiotics are living microorganisms with probiotic activity, and are fed to the host intestinal flora when fed to humans and animals in the form of dried cells or effective products in the form of single or complex strains. Can have a beneficial effect. It is the first to be such a probiotic microorganism, confirming whether it can survive in the intestine through the stomach while being less affected by gastric juice and bile, and can settle and survive in the intestine This should have a beneficial effect on the host gut. Therefore, it must have acid resistance to gastric acid, bile acid resistance to bile acids and adhesion to intestinal epithelial cells. Second, in order to be a probiotic microorganism, there must be no problem with the safety of the microorganism, and in this connection generally gelatin liquefaction test, phenylalanine deamination production test, ammonia production Tests, hemolytic experimental tests, etc. are performed. Lactobacillus plantarum CJLP56 according to the present invention not only has excellent acid resistance, bile acid resistance to bile acid intestinal epithelial cells, but also gelatin liquefaction test, phenylalanine deamine production test and ammonia production test It was found to be safe because it was confirmed by α-hemolysis, which was determined to be unrelated to pathogenicity in experimental hemolysis.

本発明にともなうラクトバシルロス・プランタラムCJLP56は、耐酸性、耐胆汁酸性および腸皮細胞付着性が非常に優秀で整腸効果が予想される。したがって、本発明は、他の側面において、ラクトバシルロス・プランタラムCJLP56を含む腸疾患の予防または治療用組成物を提供する。   Lactobacillus plantarum CJLP56 according to the present invention is extremely excellent in acid resistance, bile acid resistance and enteric cell adhesion, and is expected to have an intestinal regulating effect. Therefore, in another aspect, the present invention provides a composition for preventing or treating intestinal diseases comprising Lactobacillus plantarum CJLP56.

前記本発明にともなう微生物を含む腸疾患の予防または治療用組成物は、人を含んだ哺乳動の腸疾患の予防または治療に利用されるし、好ましくは牛、馬、豚のような家畜を含む。前記'腸疾患'では腸ために細菌感染および炎症性腸疾患を全部含んで、例えば病原性微生物(大腸菌、サルモネラ、クルロ ストゥリディウムなど)による感染性下痢、胃腸炎、炎症性腸疾患、神経性腸炎症候群、小腸微生物過成長症、腸急性下痢などを含むが、これに限定されるのではない。前記腸疾患治療用組成物に含まれるラクトバシルロス・プランタラムCJLP56は、生菌体または死菌体として存在することもできるが、生菌体として存在することが好ましい。一般的に生菌体は、腸内細菌総の異常発効によって引き起こされる諸般症状を治療して改善する効果があって人および動物に投与すれば腸内の消化管壁に密集、定着して有害菌が定着できないようにする作用をしながら、乳酸を生成して腸内のpHを低くして有害細菌が増殖できないようにする。また、投与された生菌体は、バクテリオシン(bacteriocin)と過酸化物を生成して病院菌の増殖を抑制して栄養分の吸収を担当する腸絨毛の活動を助ける。この他にも、栄養素の吸収と利用を助ける物質を生成して、動物において飼料要求率を改善させて、病原菌が生成する毒性物質を中和する物質を生成したりもする。   The composition for preventing or treating enteric diseases containing microorganisms according to the present invention is used for preventing or treating enteric diseases of mammals including humans, and preferably for domestic animals such as cattle, horses and pigs. Including. The above “intestinal diseases” include all bacterial infections and inflammatory bowel diseases because of intestines, such as infectious diarrhea, gastroenteritis, inflammatory bowel diseases, neurological enteritis caused by pathogenic microorganisms (e.g., E. coli, Salmonella, Kurosuturidium) Including, but not limited to, syndrome, small intestinal microbial overgrowth, acute intestinal diarrhea, and the like. Lactobacillus plantarum CJLP56 contained in the composition for treating intestinal diseases can be present as living cells or dead cells, but is preferably present as living cells. In general, viable cells are effective in treating and ameliorating various symptoms caused by abnormal effects of total intestinal bacteria. While preventing the fungus from colonizing, it produces lactic acid and lowers the pH in the intestine so that harmful bacteria cannot grow. In addition, the administered viable cells produce bacteriocin and peroxide to suppress the growth of hospital bacteria and help the activity of intestinal villi responsible for absorption of nutrients. In addition to this, substances that aid in the absorption and utilization of nutrients are generated to improve the feed demand rate in animals and to generate substances that neutralize toxic substances produced by pathogenic bacteria.

前記本発明の腸疾患の予防または治療用組成物の投与方法は、特に限定されるのではないか、經口で投与することが好ましい。投与量は、腸疾患の種類、疾患の程度、年齢、性別、人種、治療または予防目的などにより変われるが、一般的に成人を基準として一日に1千万匹で1000億匹を投与することができる。   The method for administering the composition for preventing or treating intestinal diseases of the present invention is not particularly limited, but it is preferably administered through the mouth. The dose varies depending on the type of intestinal disease, the degree of disease, age, sex, race, therapeutic or preventive purpose, etc. can do.

また、本発明のラクトバシルロス・プランタラムCJLP56は、整腸効果その他にも従来の乳酸菌に比べて顕著に優秀な免疫増強効果を持つ。ラクトバシルロス・プランタラムCJLP56は、脾臟細胞(splenocyte)でTh1反応を誘導するIL-12の生成を増加させて、Th2反応を誘導するIL-4の生成を抑制する。また、抗原提示細胞としてT細胞免疫反応を調節するマクロファージおよび樹状細胞のような免疫調節関連した細胞を刺激してTh0リンパ球のTh1リンパ球での分化を誘導するサイトキンの生成を促進させてTh2反応の過剰によるTh1/Th2不均衡を調節できる免疫調節力があると確認された。ラクトバシルロス・プランタラムCJLP56の免疫増強効果に対してより具体的に説明すれば次のようだ。   In addition, the Lactobacillus plantarum CJLP56 of the present invention has a remarkably superior immune enhancing effect in addition to the intestinal regulating effect and other conventional lactic acid bacteria. Lactobacillus plantarum CJLP56 increases the production of IL-12 that induces a Th1 response in splenocytes and suppresses the production of IL-4 that induces a Th2 response. In addition, it stimulates immunomodulation-related cells such as macrophages and dendritic cells that regulate T cell immune responses as antigen presenting cells, and promotes the production of cytokin that induces differentiation of Th0 lymphocytes into Th1 lymphocytes. Thus, it was confirmed that there is an immunoregulatory force capable of regulating the Th1 / Th2 imbalance caused by excessive Th2 response. A more specific explanation of the immune enhancement effect of Lactobacillus plantarum CJLP56 is as follows.

ラクトバシルロス・プランタラムCJLP56は、陰性対照群に比べて、卵アルブミン(OVA)を投与してTh2反応で偏向したマウスの脾臟細胞(spleenocyte)に処理時Th1反応誘導サイトキンのIL-12を5.8-8.4倍高く生成したし、Th2反応誘導サイトキンのIL-4の生成を10.7-12.9%水準で抑制したし、これは他の典型的な乳酸菌Lactobacillus rhamnosusGG(KCTC 5033)、Lactobacillus casei (KCTC 3109) 、Lactobacillus sakei CJLS118(KCTC 13416)に比べて顕著に優秀だと確認された。したがって、ラクトバシルロス・プランタラムCJLP56は、Th2反応を抑制してTh1反応を促進して、Th2反応の過剰によるTh1/Th2不均衡を調節する免疫調節力があるといえる。   Compared to the negative control group, Lactobacillus plantarum CJLP56 was treated with Th1 response-inducing cytokin IL-12 when treated with ovalbumin (OVA) and spleenocytes of mice that were biased by Th2 response. It produced 5.8-8.4 times higher, and suppressed IL-4 production of Th2 reaction-induced cytokin at the level of 10.7-12.9%, which is another typical lactic acid bacterium Lactobacillus rhamnosusGG (KCTC 5033), Lactobacillus casei (KCTC 3109), Lactobacillus sakei CJLS118 (KCTC 13416) was confirmed to be significantly superior. Therefore, Lactobacillus plantarum CJLP56 can be said to have immunomodulatory ability to suppress Th2 response and promote Th1 response, and to regulate Th1 / Th2 imbalance due to excessive Th2 response.

また、ラクトバシルロス・プランタラムCJLP56(Lactobacillus plantarum CJLP56)をマクロファージ細胞株RAW264.7および樹状細胞JAWSIIに処理した結果、乳酸菌数が増加するということによってマクロファージ細胞株を刺激して免疫反応を増強させるということが確認された。マクロファージ細胞株RAW264.7と受賞細胞細胞株JAWSIIにラクトバシルロス・プランタラムCJLP56を処理した結果、Th1分化誘導サイトキンIL-12、IL-18生成を促進してTh1分化誘導抑制サイトキンのIL-10はIL-12の生成量に比べて相対的に少なく生成することによってTh1分化誘導を促進すると確認された。したがって、このような実験結果からもラクトバシルロス・プランタラムCJLP56は、Th2反応を抑制してTh1反応を促進して、Th2反応の過剰によるTh1/Th2不均衡を調節する免疫調節力があるといえる。   In addition, Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) was treated with macrophage cell line RAW264.7 and dendritic cell JAWSII, resulting in an increase in the number of lactic acid bacteria. It was confirmed that Macrophage cell line RAW264.7 and award cell line JAWSII were treated with Lactobacillus plantarum CJLP56, resulting in Th1 differentiation-inducing cytokin IL-12 and IL-18 production and Th1 differentiation-inducing cytokin IL -10 was confirmed to promote the induction of Th1 differentiation by producing relatively less than the amount of IL-12 produced. Therefore, even from these experimental results, Lactobacillus plantarum CJLP56 has an immunomodulatory ability to suppress Th2 response, promote Th1 response, and regulate Th1 / Th2 imbalance due to excessive Th2 response I can say that.

IL-4は、Th2細胞で由来して特異的に細胞免疫反応の中枢的な役割をしてTh1細胞のサイトキンのIL-12の生産を抑制する抗炎症性サイトキン(anti-inflammatory cytokine)機能を持つ。近ごろアトピー皮膚炎患者の末梢血液と皮膚病変にはIL-4,IL-5等を主に生産するTh2細胞が相対的に増加するという事実が知られている(非特許文献10)。したがって、Th2反応過剰によるTh1/Th2の不均衡は、アトピーのような疾患を誘発する。また、先立って調べたように、Th1反応とTh2反応の中の一つが過剰や他の一つが不足すれば病気が引き起こされて、Th1反応が低下してTh2反応が増加時、癌、アトピー、アレルギーおよび自家免疫疾患が引き起こされると知られている(非特許文献11)。したがって、本発明にともなうラクトバシルロス・プランタラムCJLP56は、免疫調節と関連したTh1細胞、Th2細胞、マクロパジおよび樹状細胞が生産するサイトキンを調節してTh2反応過剰によるTh1/Th2の不均衡を調節することによって、アトピー、アレルギーのような病気に対し効果的に作用する可能性があることを期待できるだけでなく、癌および自家免疫疾患の予防または治療にも効果があると期待される。   IL-4 is an anti-inflammatory cytokine that is derived from Th2 cells and specifically plays a pivotal role in the cellular immune response and suppresses IL-12 production of Th1 cell cytokin Has function. Recently, it is known that Th2 cells mainly producing IL-4, IL-5 and the like are relatively increased in peripheral blood and skin lesions of atopic dermatitis patients (Non-patent Document 10). Thus, Th1 / Th2 imbalance due to excessive Th2 response induces diseases such as atopy. In addition, as previously examined, when one of the Th1 and Th2 reactions is excessive or one of the other is insufficient, the disease is caused, and when the Th1 response decreases and the Th2 response increases, cancer, atopy, It is known that allergies and autoimmune diseases are caused (Non-patent Document 11). Thus, Lactobacillus plantarum CJLP56 in accordance with the present invention regulates Th1 / Th2 imbalance due to Th2 hyperresponsiveness by regulating Thkin, Th2 cells, macropaj and dendritic cells produced by immunomodulation. It is expected not only that it may effectively act against diseases such as atopy and allergy, but is also expected to be effective in the prevention or treatment of cancer and autoimmune diseases.

したがって、本発明は、他の側面において、ラクトバシルロス・プランタラムCJLP56を含む免疫増強用組成物を提供する。本発明にともなう免疫増強用組成物は、ラクトバシルロス・プランタラムCJLP56が乳酸菌として前記従来技術で説明したことのような一般的な乳酸菌の免疫増強作用効果によって免疫増強効果がある。特に、本発明にともなう前記免疫増強用組成物は、下記実施例で立証された通りラクトバシルロス・プランタラムCJLP56がTh1反応の促進効果を持ってTh2反応過剰によるTh1/Th2の不均衡を調節する効果があるので、Th2反応過剰によるTh1/Th2の不均衡に誘発される疾患の予防または治療に効果がある。したがって、本発明にともなう前記免疫増強用組成物は、アトピー、アレルギー、癌および自家免疫疾患の予防または治療に効果的に使われることができる。前記自家免疫疾患では喘息、枯草熱などを含むが、これに限定されるのではない。   Therefore, in another aspect, the present invention provides an immunopotentiating composition comprising Lactobacillus plantarum CJLP56. The composition for enhancing immunity according to the present invention has an immune enhancing effect due to the immune enhancing effect of a general lactic acid bacterium as described in the above-mentioned prior art as Lactobacillus plantarum CJLP56 as a lactic acid bacterium. In particular, in the composition for enhancing immunity according to the present invention, as demonstrated in the Examples below, Lactobacillus plantarum CJLP56 has an effect of promoting Th1 reaction and regulates Th1 / Th2 imbalance due to excessive Th2 reaction Therefore, it is effective for the prevention or treatment of diseases induced by Th1 / Th2 imbalance caused by excessive Th2 response. Therefore, the composition for enhancing immunity according to the present invention can be effectively used for the prevention or treatment of atopy, allergy, cancer and autoimmune diseases. The autoimmune diseases include, but are not limited to, asthma and hay fever.

前記本発明の免疫増強用組成物の投与方法は、特に限定されるのではないか、經口で投与することが好ましい。投与量は、免疫増強が必要な疾患の種類、疾患の程度、年齢、性別、人種、治療または予防目的などにより変われるが、一般的に成人を基準として一日に1千万匹で1000億匹を投与することができる。   The method for administering the composition for enhancing immunity of the present invention is not particularly limited, and it is preferable to administer the composition through the mouth. The dose varies depending on the type of disease that requires immune enhancement, the degree of disease, age, sex, race, therapeutic or preventive purpose, etc. One hundred million animals can be administered.

前記本発明にともなうラクトバシルロス・プランタラムCJLP56を含む腸疾患の予防または治療用組成物および免疫増強用組成物は、安全性が立証された乳酸菌を含むので副作用などの憂慮なしで医薬品、食品、化粧品、飼料、または飼料添加剤として利用されることができる。   The composition for preventing or treating intestinal diseases and the composition for enhancing immunity containing Lactobacillus plantarum CJLP56 according to the present invention contains a lactic acid bacterium that has been proven to be safe, so that there are no side effects and other concerns. It can be used as a cosmetic, feed, or feed additive.

前記本発明にともなう組成物が医薬品として利用される場合には、当該技術分野に公示されている通常の薬剤学的剤形で製剤化されることができる。前記医薬品は、好ましくは經口剤形を製剤化されるし、例えば液体額剤、懸濁剤、散剤、顆粒剤、精剤、カプセル剤、丸剤またはエックス剤のような経口投与用剤形で製剤化されることができる。   When the composition according to the present invention is used as a medicine, it can be formulated in a usual pharmaceutical dosage form publicly disclosed in the technical field. The medicament is preferably formulated in a mouthpiece dosage form, for example liquid dosage forms, suspensions, powders, granules, essences, capsules, pills or X-forms. Can be formulated.

前記それぞれの剤形で製剤化詩、それぞれの剤形の製造に必要な薬剤学的に許容可能な担体または添加剤を付加して製造することができる。代表的に経口投与用剤形で製剤化詩前記担体として希釈剤、滑澤剤、結合剤、崩解剤、甘美剤、安定剤および防腐剤中で1種以上を選択して使えるし、添加剤では香料、ビタミン類および抗酸化剤中で1種以上を選択して使うことができる。   Each of the dosage forms can be prepared by adding a formulation poem and a pharmaceutically acceptable carrier or additive necessary for the production of each dosage form. Typically formulated as a dosage form for oral administration. As a carrier, one or more can be selected and used as diluent, lubricant, binder, disintegrant, sweetener, stabilizer and preservative. One or more kinds of fragrances, vitamins, and antioxidants can be selected and used.

前記担体および添加剤は、薬剤学的に許容可能になることは全部可能で、具体的に希釈剤では乳糖、とうもろこし澱粉、大豆油、米晶質セルロース、またはマンニトル、滑澤剤ではステアリン酸マグネシウムまたはタルク、結合剤ではポルリビニルピロルリドンまたはヒドロックシプロフィールセルロースが好ましい。また、崩解剤ではカルボクシメティルセルロースカルシウム、澱粉グリコール酸ナトリウム、ポルラクリルリンカルリュムまたはクロスポビドン、甘美剤では白糖、果糖、ソルビトル、または、アスパルテーム、安定剤ではカルボクシメティルセルロースナトゥリュム、β-サイクルロデクストゥリン、白斑、またはチャンタンゴム、防腐剤ではパラオクシ安息香酸メティル、パラオキシ安息香酸プロフィール、または、ソルビン酸カリウムが好ましい。   The carriers and additives can all be pharmaceutically acceptable, specifically lactose, corn starch, soybean oil, rice crystalline cellulose, or mannitol for diluents, magnesium stearate for lubricants. Alternatively, talc and binder are preferably poly (vinylpyrrolidone) or hydroxyl profile cellulose. Also, disintegrants are carboxymethyl cellulose calcium, sodium starch glycolate, porlacryl phosphorus calcium or crospovidone, sweeteners are sucrose, fructose, sorbitol or aspartame, and stabilizers are carboxymethyl cellulose cellulose. Ryumu, β-cyclerodexturin, vitiligo, or Chantan gum, and as preservatives, methyl oxybenzoate, paraoxybenzoic acid profile, or potassium sorbate is preferred.

また、前記成分その他にも公示の添加剤として味覚を注ぐために、梅の実香り、レモン香り、パイナップル香り、ハブ香りなどの天然香料、天然果汁、クロロフィリン、フラボノイドなどの天然色素、果糖、蜂蜜、糖アルコール、砂糖と同じ甘美成分、または、枸櫞酸、枸櫞酸ナトリウムと同じ酸味剤を混合して使うこともできる。   In addition to natural ingredients such as plum scent, lemon scent, pineapple scent, hub scent, natural fruit juice, chlorophyllin, flavonoids, natural pigments, fructose, honey, Sugar alcohol, the same sweetening ingredient as sugar, or the same sour agent as oxalic acid and sodium oxalate can be used in combination.

このような製剤化方法および製剤化時必要な担体および添加剤に対しては非特許文献11に詳細に記載されている。   Non-patent document 11 describes in detail such formulation methods and carriers and additives necessary for formulation.

前記本発明にともなう組成物は、食品として利用されることができる。前記食品は健康機能食品それだけでなく、人間が広く通常的に毎日摂取する一般的な食品を含むことだ。健康機能食品として利用される場合、食品学的に許容可能な担体または添加剤とともに当該技術分野に公示されている通常の健康機能食品の剤形で製剤化されることができる。前記健康機能食品は、例えば酸剤、顆粒剤、精剤、カプセル剤、懸濁液、エマルジョン、シロップ剤、液剤、エックス剤、茶、ジェリー、または飲み物などで製造されることができる。前記食品学的に許容可能な担体または添加剤は、製造しようと思う剤形の製造に当該技術分野で使用可能になることで公示されている任意の担体または添加剤が利用されることができる。   The composition according to the present invention can be used as a food. These foods include not only health-functional foods, but also general foods that are widely and regularly consumed every day by humans. When utilized as a health functional food, it can be formulated in the form of a normal health functional food advertised in the art together with a pharmaceutically acceptable carrier or additive. The health functional food can be produced, for example, as an acid agent, granule, essential agent, capsule, suspension, emulsion, syrup, solution, X agent, tea, jelly, or drink. As the food-acceptable carrier or additive, any carrier or additive publicly known to be usable in the art for the production of a dosage form to be manufactured can be used. .

前記本発明にともなう組成物は、アトピーの予防または治療効果を持つので、化粧品として利用されることもできる。前記本発明にともなう組成物が化粧品として利用される場合には当該化粧品技術分野に公示されている通常の剤形の多様な化粧品で製造されることができる。前記それぞれの剤形で製剤化詩、それぞれの剤形の製造に必要な化粧品の製造に許容可能な担体または添加剤を付加して製造することができる。   Since the composition according to the present invention has an effect of preventing or treating atopy, it can also be used as a cosmetic. When the composition according to the present invention is used as a cosmetic, it can be manufactured with various cosmetics in a usual dosage form advertised in the cosmetic technical field. Each of the dosage forms can be prepared by adding a formulation poem and a carrier or additive acceptable for the production of cosmetics necessary for the production of each dosage form.

前記本発明にともなう組成物は、飼料添加剤または、飼料として利用されることができる。   The composition according to the present invention can be used as a feed additive or a feed.

飼料添加剤として利用される場合、前記組成物は、20ないし90%高濃縮液や粉末または顆粒形態で製造されることができる。前記飼料添加剤は、枸櫞酸、フマル酸、アディピック酸、乳酸、リンゴ酸などの有機酸や燐酸ナトリウム、燐酸カリウム、酸性疲労燐酸炎、ポリ燐酸炎(重合燐酸炎)等の燐酸炎や、ポリフェノール、カテキン、α-トコフェロール、ローズマリー抽出物、ビタミンC、緑茶抽出物、甘草抽出物、キトサン、タンニン酸、ピティン酸などの天然抗酸化剤中どれ一つまたは一つ以上を追加で含むことができる。飼料として利用される場合、前記組成物は、通常の飼料形態で製剤化されるし、通常の飼料成分を共に含むことができる。   When used as a feed additive, the composition can be produced in the form of a 20 to 90% highly concentrated solution, powder or granules. The feed additive is an organic acid such as oxalic acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphoric acid flame such as sodium phosphate, potassium phosphate, acidic fatigue phosphoric acid flame, polyphosphoric acid flame (polymerized phosphoric acid flame), Contains one or more additional natural antioxidants such as polyphenols, catechins, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, pitinic acid Can do. When used as a feed, the composition is formulated in a normal feed form and can include both normal feed ingredients.

前記飼料添加剤および飼料は、穀物、例をあげれば粉砕または破砕されたコムギ、燕麦、麦、とうもろこしおよび米;植物性蛋白質飼料、例をあげればアブラナ、豆、およびひまわりを主成分でする飼料;動物性蛋白質飼料、例をあげれば血分、肉粉、骨粉および魚分;糖分および乳製品、例をあげれば各種粉乳および乳状粉末で成り立つ乾燥成分などをさらに含めるし、その他にも栄養補充剤、消化および吸収向上剤、成長促進剤などをさらに含むことができる。   The feed additive and the feed are cereals such as wheat, oats, wheat, corn and rice pulverized or crushed, for example; vegetable protein feeds such as rape, beans, and sunflower. ; Animal protein feed, blood, meat meal, bone meal and fish for example; sugar and dairy products, dry ingredients made up of various milk powder and milk powder for example, and other nutritional supplements , Digestion and absorption enhancers, growth promoters and the like.

前記飼料添加剤は、動物に単独で投与したり食用担体中で他の飼料添加剤と組み合わせて投与することもできる。また、前記飼料添加剤は、トップドレッシングとしてまたはこれらを動物飼料に直接混合したりまたは飼料と別途の經口剤形で容易に動物に投与することができる。前記飼料添加剤を動物飼料と別に投与する場合、当該技術分野に良く知られた通り薬剤学的に許容可能な食用担体と組み合わせて、直ちに放出または徐放性剤形で製造することができる。このような食用担体は、固体または液体、例えばとうもろこし澱粉、ラクトース、スクロオス、豆フレーク、落花生油、オリーブ油、胡麻油およびプロピレングリコールでありうる。固体担体が使われる場合、飼料添加剤は、精剤、カプセル剤、酸剤、トローチ剤または含糖精剤または、未分散性形態のトップドレッシングでありうる。液体担体が使われる場合、飼料添加剤は、ゼラチン軟質カプセル剤、またはシロップ剤や懸濁液、エマルジョン剤、または溶液剤の剤形でありうる。   The feed additive can be administered to animals alone or in combination with other feed additives in an edible carrier. In addition, the feed additive can be easily administered to animals as a top dressing, directly mixed with animal feed, or in a mouthpiece form separate from the feed. When the feed additive is administered separately from animal feed, it can be produced in immediate release or sustained release dosage form in combination with a pharmaceutically acceptable edible carrier as is well known in the art. Such edible carriers can be solid or liquid, such as corn starch, lactose, scroos, bean flakes, peanut oil, olive oil, sesame oil and propylene glycol. If a solid carrier is used, the feed additive can be a essence, capsule, acid, troche or sugar-containing essence or an undispersed form of top dressing. When a liquid carrier is used, the feed additive can be a gelatin soft capsule, or a syrup or suspension, emulsion, or solution dosage form.

前記飼料は、動物の食餌欲求を充足させるのに通常的に使われる任意の蛋白質-含有有機穀粉を含むことができる。このような蛋白質-含有穀粉は、通常的にとうもろこし、豆穀粉、またはとうもろこし/豆穀粉ミックスで主に構成されている。   The feed can include any protein-containing organic flour normally used to satisfy an animal's dietary needs. Such protein-containing flours are usually composed primarily of corn, bean flour, or a corn / bean flour mix.

また、前記飼料添加剤および飼料は、補助剤、例えば保存剤、安定化剤、湿潤剤または乳化剤、溶液促進剤などを含有することができる。前記飼料添加剤は、浸透、噴霧または、混合して動物の飼料に添加して利用されることができる。   In addition, the feed additive and the feed may contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifiers, solution accelerators, and the like. The feed additive can be used by being added to animal feed after being permeated, sprayed or mixed.

本発明の飼料または飼料添加剤は、哺乳類、家禽および魚類を含む多数の動物食餌に適用することができる。前記哺乳類として豚、牛、羊、ヤギ、実験用設置動物および実験用設置動物それだけでなく、愛玩動物(例:犬、猫)等に使えるし、前記家禽類として鶏、七面鳥、鴨、ガチョウ、キジおよびウズラなどにも使用できて、前記魚類として鱒などに利用されるが、これに限定されるのではない。   The feed or feed additive of the present invention can be applied to many animal diets including mammals, poultry and fish. The mammal can be used for pigs, cows, sheep, goats, laboratory animals and laboratory animals, as well as pets (e.g. dogs, cats), etc., and the poultry can be chickens, turkeys, duck, geese, It can also be used for pheasants, quails, and the like, but is not limited to this.

先立って説明した通り、本発明にともなう新規乳酸菌ラクトバシルロス・プランタラムCJLP56は、プロバイオティクスとして耐酸性、耐胆汁酸性および腸上皮細胞付着性が非常に優秀で整腸効果を持って、Th1反応を促進する効果があってTh2反応過剰によるTh1/Th2の不均衡を調節する効果がある。したがって、本発明にともなう新規乳酸菌ラクトバシルロス・プランタラムCJLP56は、腸疾患治療用組成物および免疫増強用組成物として利用されるし、特にTh2反応過剰によるTh1/Th2の不均衡に誘発される疾患の予防または治療に効果的だ。   As described above, the novel lactic acid bacterium Lactobacillus plantarum CJLP56 according to the present invention has excellent acid resistance, bile acid resistance and intestinal epithelial cell adhesion as a probiotic, and has an intestinal regulating effect. It has the effect of promoting the reaction and the effect of regulating the Th1 / Th2 imbalance due to excessive Th2 reaction. Therefore, the novel lactic acid bacterium Lactobacillus plantarum CJLP56 according to the present invention is used as a composition for treating intestinal diseases and a composition for enhancing immunity, and particularly induced by Th1 / Th2 imbalance due to excessive Th2 response. It is effective in preventing or treating diseases.

図1は、ラクトバシルロス・プランタラムCJLP56の耐酸性実験結果を示すグラフである。FIG. 1 is a graph showing the acid resistance test results of Lactobacillus plantarum CJLP56. 図2は、ラクトバシルロス・プランタラムCJLP56の耐胆汁酸性実験結果を示すグラフである。FIG. 2 is a graph showing the bile acid resistance experimental results of Lactobacillus plantarum CJLP56. 図3は、ラクトバシルロス・プランタラムCJLP56の腸上皮細胞付着能実験結果を示すグラフである。FIG. 3 is a graph showing the intestinal epithelial cell adhesion ability test results of Lactobacillus plantarum CJLP56. 図4は、ラクトバシルロス・プランタラムCJLP56菌株を卵アルブミンを投与してTh2反応で偏向したマウスの脾臟細胞に処理した後Th1反応誘導サイトキンのIL-12の濃度を測定した結果を他の乳酸菌と比較して示すグラフである。FIG. 4 shows the results of measurement of IL-12 concentration of Th1 reaction-induced cytokin after treatment of Lactobacillus plantarum strain CJLP56 with spleen splenocytes of mice that were administered ovalbumin and biased by Th2 reaction. It is a graph shown in comparison with lactic acid bacteria. 図5は、ラクトバシルロス・プランタラムCJLP56菌株を卵アルブミンを投与してTh2反応で偏向したマウスの脾臟細胞に処理した後Th2反応誘導サイトキンのIL-4の濃度を測定した結果を他の乳酸菌と比較して示すグラフである。FIG. 5 shows the results of measurement of IL-4 concentration of Th2 reaction-induced cytokin after treatment of Lactobacillus plantarum strain CJLP56 strain with spleen sputum cells that were administered ovalbumin and biased by Th2 reaction. It is a graph shown in comparison with lactic acid bacteria. 図6は、ラクトバシルロス・プランタラムCJLP56菌株をマクロファージ細胞株RAW264.7に処理した後、IL-12およびIL-10の濃度をELISAで測定した結果を他の乳酸菌と比較して示すグラフである。FIG. 6 is a graph showing the results of measuring the concentration of IL-12 and IL-10 by ELISA after treating the Lactobacillus plantarum CJLP56 strain with the macrophage cell line RAW264.7 and comparing with other lactic acid bacteria. is there. 図7は、ラクトバシルロス・プランタラムCJLP56菌株を樹状細胞細胞株JAWSIIに処理した後、IL-12およびIL-10の濃度をELISAで測定した結果を他の乳酸菌と比較して示すグラフである。FIG. 7 is a graph showing the results obtained by treating Lactobacillus plantarum CJLP56 strain with dendritic cell line JAWSII and then measuring the concentrations of IL-12 and IL-10 by ELISA in comparison with other lactic acid bacteria. is there. 図8は、ラクトバシルロス・プランタラムCJLP56菌株をマクロファージ細胞株RAW264.7に処理した後、IL-12p40およびIL-18 mRNAの発現をRT-PCRで測定した結果を他の乳酸菌と比較して示すグラフである。FIG. 8 shows the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating the Lactobacillus plantarum CJLP56 strain with the macrophage cell line RAW264.7 and comparing it with other lactic acid bacteria. It is a graph to show. 図9は、ラクトバシルロス・プランタラムCJLP56菌株を樹状細胞細胞株JAWSIIに処理した後、IL-12p40およびIL-18 mRNAの発現をRT-PCRで測定した結果を他の乳酸菌と比較して示すグラフである。FIG. 9 shows the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating the Lactobacillus plantarum CJLP56 strain with the dendritic cell line JAWSII and comparing it with other lactic acid bacteria. It is a graph to show.

以下、本発明を下記実施例によってより一層具体的に説明する。しかし、これら実施例は本発明に対する理解を助けるためのものだけ、どんな意味でも本発明の範囲がこれらによって制限されるのではない。   Hereinafter, the present invention will be described more specifically with reference to the following examples. However, these examples are only for helping understanding of the present invention and do not limit the scope of the present invention in any way.

実験例1:微生物ラクトバシルロス・プランタラムCJLP56菌株の分離および同定
キムチで分離したラクトバシルロス・プランタラムCJLP56菌株を1.5%寒天(agar)が含まれたMRS固体培地(Difco, USA)に塗抹して37℃で24時間培養した後純粋分離したことが確認された集落を白金が(loop)で取ってMRS液体培地(Difco, USA)で37℃18-24時間培養した。
Experimental Example 1: Isolation and identification of microbial Lactobacillus plantarum CJLP56 strain Lactobacillus plantarum CJLP56 strain isolated by Kimchi was smeared on MRS solid medium (Difco, USA) containing 1.5% agar (agar) The colonies that were confirmed to be purely isolated after culturing at 37 ° C. for 24 hours were taken as platinum (loop) and cultured in MRS liquid medium (Difco, USA) at 37 ° C. for 18-24 hours.

その次に、CJLP56菌株の形態および生理学的特性をKimなど(非特許文献12)の方法とAPI50CHおよびAPI50CH Lキット(バイオメリオ社製品)を使って決めた。その結果確認されたCJLP56菌株の形態および生理学的特性を前記表1に整理した。   Next, the morphology and physiological characteristics of the CJLP56 strain were determined using the method of Kim et al. (Non-patent Document 12) and the API50CH and API50CH L kits (Biomerio product). The morphology and physiological characteristics of the CJLP56 strain confirmed as a result are summarized in Table 1 above.

また、乳酸菌同定および分類のための16S rRNA遺伝子の塩基配列を分析した。16S rRNA遺伝子の塩基配列決定および分析は、Kimなど(非特許文献13)の方法を使った。その結果確認されたCJLP56の16S rRNA遺伝子の塩基配列は、序列目録に記載した(SEQ ID NO:1) 。   The base sequence of 16S rRNA gene for lactic acid bacteria identification and classification was analyzed. The method of Kim et al. (Non-patent Document 13) was used to determine and analyze the base sequence of the 16S rRNA gene. The nucleotide sequence of the 16S rRNA gene of CJLP56 confirmed as a result was listed in the sequence listing (SEQ ID NO: 1).

ラクトバシルロス・プランタラムCJLP56菌株は、16S rRNA塩基配列分析結果ラクトバシルロス・プランタラム標準菌株(Lactobacillus plantarum NBRC 15891T、GenBank accession number AB326351)と最も高い相同性(99.9%)を現わしてラクトバシルロス・プランタラム(Lactobacillus plantarm)で同定して、ラクトバシルロス・プランタラムCJLP56で命名したし、生命工学研究院遺伝子銀行に2008年10月16日付で寄託した(受託番号KCTC 11402 BP)。   Lactobacillus plantarum CJLP56 strain showed the highest homology (99.9%) with Lactobacillus plantarum NBRC 15891T, GenBank accession number AB326351 as a result of 16S rRNA nucleotide sequence analysis. It was identified with Lactobacillus plantarm, named Lactobacillus plantarum CJLP56, and deposited with the Biotechnology Institute Genebank on October 16, 2008 (accession number KCTC 11402 BP).

実験例2:ラクトバシルロス・プランタラムCJLP56菌株の人工胃液での耐酸性実験および人工胆汁での耐胆汁性実験
人工胃液での耐酸性実験は、Kobayashiなど(非特許文献14)の実験を変形して製造した人工胃液を使って進行したし、具体的に人工胃液は、MRS液体培地を1N HClでpH 2.5になるべく調整してペブシンを1000ユニット/mlになるべく添加した後滅菌して製造した。
Experimental Example 2: Lactobacillus plantarum CJLP56 strain acid resistance experiment with artificial gastric juice and bile resistance experiment with artificial bile Acid resistance experiment with artificial gastric juice is a modification of the experiment of Kobayashi et al. The artificial gastric juice was prepared using the prepared artificial gastric juice. Specifically, the artificial gastric juice was prepared by sterilizing the MRS liquid medium with 1N HCl to pH 2.5 and adding pebcine to 1000 units / ml. .

前記実施例1から分離および同定されたラクトバシルロス・プランタラムCJLP56をMRS液体培地で37℃、18時間の間培養した菌体を遠心分離して乳酸菌主を沈殿させてまた滅菌食塩水(0.85%NaCl)で2回洗浄した後菌体懸濁液を対照群培地と人工胃液に各々約107cfu/ml水準で接種させて37℃で培養させながら接種後0および3時間後に生存菌数を測定した。総菌数は、KH2PO4、Na2HPO、L-システイン、HCl、Tween 80等が含まれた燐酸緩衝液(pH 6.8)で10倍数希釈して測定した。 Lactobacillus plantarum CJLP56 isolated and identified from Example 1 above was cultured in an MRS liquid medium at 37 ° C. for 18 hours to centrifuge the lactic acid bacteria to precipitate, and sterile saline (0.85 After rinsing twice with (NaCl), the bacterial cell suspension was inoculated into the control medium and artificial gastric juice at a level of about 10 7 cfu / ml, and cultured at 37 ° C. Was measured. The total number of bacteria was measured by diluting 10 times with a phosphate buffer (pH 6.8) containing KH 2 PO 4, Na 2 HPO, L-cysteine, HCl, Tween 80 and the like.

人工胆汁での耐胆汁性実験は、Caseyなど(非特許文献15)の方法により進行したし前記耐酸性評価で使われたMRS液体培地に黄牛胆汁を0.3%添加した後、前記耐酸性評価方法と同じ方法でして乳酸菌接種後0時間、12時間、24時間後に生存菌数を測定した。   The bile resistance experiment with artificial bile progressed by the method of Casey et al. (Non-patent Document 15). After 0.3% of bull bile was added to the MRS liquid medium used in the acid resistance evaluation, the acid resistance evaluation method The number of viable bacteria was measured after 0, 12, and 24 hours after inoculation with lactic acid bacteria.

前記耐酸性評価および耐胆汁酸性評価皆で典型的な乳酸菌のLactobacillus c asei(KCTC 3109)、Lactobacillus sakei CJLS118(KCTC 13416)、およびLactoba ilus rhamnosus GG(KCTC 5033)に対しても同一に比較実験を遂行した。   The same comparative experiment was conducted for the above-mentioned acid resistance evaluation and bile acid resistance evaluation for Lactobacillus casei (KCTC 3109), Lactobacillus sakei CJLS118 (KCTC 13416), and Lactoba ilus rhamnosus GG (KCTC 5033). Carried out.

その結果を図1および図2に示した。図1はラクトバシルロス・プランタラムCJLP56の耐酸性実験結果を示したグラフである。図2はラクトバシルロス・プランタラムCJLP56の耐胆汁酸性実験結果を示したグラフである。   The results are shown in FIG. 1 and FIG. FIG. 1 is a graph showing the results of acid resistance experiments of Lactobacillus plantarum CJLP56. FIG. 2 is a graph showing the results of bile acid resistance experiment of Lactobacillus plantarum CJLP56.

図1および図2の結果によれば、ラクトバシルロス・プランタラムCJLP56は、比較実験した他の乳酸菌皆に比べて同等以上の耐酸性および耐胆汁酸性を持つことが明らかになった。これは本発明にともなう新規菌株が体内で胃液の影響を受けないで腸まで到達することができるし腸内では胆汁の影響を受けないで生存する可能性があることを現わす。   From the results of FIGS. 1 and 2, it was revealed that Lactobacillus plantarum CJLP56 has acid resistance and bile acid resistance equal to or higher than those of all other lactic acid bacteria subjected to the comparative experiments. This indicates that the novel strain according to the present invention can reach the intestine without being affected by gastric juice in the body and can survive without being affected by bile in the intestine.

実施例3:ラクトバシルロス・プランタラムCJLP56菌株の腸上皮細胞に対する付着能実験
腸上皮細胞付着能試験のための動物細胞としてHT-29を韓国細胞株銀行(KCLB)で譲り受けて使ったし、実験方法は、キムなど(非特許文献16)とHiranoなど(非特許文献17)の方法を使った。
Example 3: Lactobacillus plantarum CJLP56 strain adhesion test for intestinal epithelial cells HT-29 was used as an animal cell for intestinal epithelial cell adhesion test and was used at the Korea Cell Line Bank (KCLB), The experimental method used was Kim et al. (Non-Patent Document 16) and Hirono et al. (Non-Patent Document 17).

HT-29細胞は、熱非活性化された10%牛胎児血清(FBS)、1% L-グルタミン、ペニシリンG(100IU/mL)、そしてストレプトマイシン(100mg/mL)が添加されたRPMI 1640(Gibco, USA)培地を利用して5%CO2存在下で37℃で培養させた。付着能実験と付着抑制能実験のためにHT-29細胞は、ウェル当1.0×105細胞/mLの数になるべく24ウェル-プレートに分株しかったし隔日で培地を交換して完全に単一層(monolayer)を形成する時まで培養して実験に使った。完全単一層を形成したHT-29細胞は、25℃のPBS緩衝溶液を利用して5回洗浄して抗生剤が添加されなかったRPMI 1640培地0.5mLを添加した。 HT-29 cells are RPMI 1640 (Gibco) supplemented with heat-inactivated 10% fetal bovine serum (FBS), 1% L-glutamine, penicillin G (100 IU / mL), and streptomycin (100 mg / mL). , USA) medium and cultured at 37 ° C. in the presence of 5% CO 2 . For adhesion and adhesion inhibition experiments, HT-29 cells were divided into 24-well plates as much as 1.0 × 10 5 cells / mL per well, and the medium was changed every other day. It was cultured until the time when a monolayer was formed and used for the experiment. HT-29 cells that formed a complete monolayer were washed 5 times using a PBS buffer solution at 25 ° C., and 0.5 mL of RPMI 1640 medium to which no antibiotic was added was added.

ラクトバシルロス・プランタラムCJLP56は、各々約1.0×109cfu/mLの濃度になるべくRPMIに懸濁した後前記ウェルプレートに接種して5% CO2存在下で37℃で2時間の間培養を実施した。培養が完了した後付着しなかった乳酸菌の除去と洗浄にともなう付着能力を確認するために3分ずつ200rpmの速度で攪拌しながらPBS緩衝溶液を使って3回洗浄を実施した。洗浄が完了した後0.2%トリプシン-EDTAを分株して付着している細胞を引き離してペプトン(peptone)数を利用して連続希釈法でMRS-あか(agar)に評判塗抹して37℃で24時間の間培養して菌数を測定した。   Lactobacillus plantarum CJLP56 was suspended in RPMI to a concentration of about 1.0 × 109 cfu / mL, inoculated into the well plate, and cultured at 37 ° C. for 2 hours in the presence of 5% CO2. . Washing was carried out 3 times using PBS buffer solution with stirring at a rate of 200 rpm for 3 minutes in order to confirm the adherence ability accompanying removal and washing of lactic acid bacteria that did not adhere after completion of the culture. After washing is completed, 0.2% trypsin-EDTA is separated to separate the attached cells, and the MRS-agar is smeared in a serial dilution method using the peptone number. The cells were cultured for 24 hours and the number of bacteria was measured.

別に、一部付着確認のために70%アルコールに一日程度漬けて完全殺菌されたカバーグラスをペットリティシュィ底に付着させた後HT-29細胞を培養して上と同量の乳酸菌を添加して実験した。洗浄によって洗い流されていかないでHT-29細胞に付着した乳酸菌株は、乾燥した後Gram染色をして光学顕微鏡で観察して、菌数を測定した。Lactobacillus sakei CJLS118、およびLactobaillus rhamnosusGG(KCTC 5033)に対しても同一に比較実験を遂行した。   Separately, in order to confirm partial adhesion, a cover glass soaked in 70% alcohol for about a day and completely sterilized is attached to the bottom of the petrity tissue, and then the HT-29 cells are cultured, and the same amount of lactic acid bacteria is added. The experiment was conducted with addition. Lactic acid strains that were not washed away by washing and adhered to HT-29 cells were dried and then stained with Gram and observed with an optical microscope to determine the number of bacteria. The same comparative experiment was performed for Lactobacillus sakei CJLS118 and Lactobaillus rhamnosusGG (KCTC 5033).

その結果を図3に示した。図3は、ラクトバシルロス・プランタラムCJLP56の腸上皮細胞付着能実験結果を示すグラフである。   The results are shown in FIG. FIG. 3 is a graph showing the intestinal epithelial cell adhesion ability test results of Lactobacillus plantarum CJLP56.

図3の結果によれば、ラクトバシルロス・プランタラムCJLP56は、プロバオティクス菌株で商業的に良く知られたLactobacillus rhamnosusGG(KCTC 5033)と Lactobacillus sakei CJLS118に比べて24時間経過後腸上皮細胞付着能が優秀なことが明らかになったし、特にLactobacillus sakei CJLS118に比べて腸上皮細胞付着能が顕著に高いことが分かった。このような結果は本発明にともなう新規菌株が腸上皮細胞に付着して腸内環境を改善する可能性があることを現わす。   According to the results in FIG. 3, Lactobacillus plantarum CJLP56 adheres to intestinal epithelial cells after 24 hours compared to Lactobacillus rhamnosusGG (KCTC 5033) and Lactobacillus sakei CJLS118, which are well known commercially in probaotic strains. It was revealed that the ability of the intestinal epithelial cells was significantly higher than that of Lactobacillus sakei CJLS118. These results indicate that the novel strain according to the present invention may adhere to intestinal epithelial cells and improve the intestinal environment.

実施例4:ラクトバシルロス・プランタラムCJLP56菌株の安全性評価
前記実施例1で分離した菌株の安全性を評価するために韓国バイオベンチャー協会団体標準で提示した安全性評価試験法により溶血現象検査、ゼラチン液化反応検査、有害代謝産物(アンモニア)生成確認、ペニンアラニンタルアミン検査を遂行した。
Example 4: Safety evaluation of Lactobacillus plantarum CJLP56 strain In order to evaluate the safety of the strain isolated in Example 1, hemolysis phenomenon test by the safety evaluation test method presented in the Korea Bioventure Association group standard Gelatin liquefaction test, harmful metabolite (ammonia) production confirmation, peninalanine taramine test were performed.

その結果を下記表2に示した。   The results are shown in Table 2 below.

Figure 2012510291
Figure 2012510291

前記結果によれば、ラクトバシルロス・プランタラムCJLP56(Lactobacillus plantarum CJLP56)は、ゼラチン液化反応、有害代謝産物(アンモニア)生成、ペニンアラニンタルアミン生成に対して陰性であり、溶血現象検査では病原性と関係がないと判定されるα-溶血で確認された。したがって、ラクトバシルロス・プランタラムCJLP56は、人体に投与できる安全な菌株だと確認された。   According to the above results, Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) is negative for gelatin liquefaction, harmful metabolite (ammonia) production, peninalanine taramine production, pathogenicity in hemolysis phenomenon test It was confirmed by α-hemolysis determined to be unrelated. Therefore, Lactobacillus plantarum CJLP56 was confirmed to be a safe strain that can be administered to the human body.

実施例5:マウス 脾臟細胞処理後IL-12生成促進力評価
ラクトバシルロス・プランタラムCJLP56(Lactobacillus plantarum CJLP56)菌株を、卵アルブミンを投与してTh2反応で偏向したマウスの脾臟細胞に処理時Th1反応誘導サイトキンのIL-12生成促進力を評価するために、Fujiwaraなど(非特許文献18)とFujiwaraなど(非特許文献19)を参考にして次の通り実験を進行した。
Example 5: Evaluation of IL-12 production promoting ability after treatment with mouse spleen sputum cells In order to evaluate the IL-12 production promoting ability of reaction-induced cytokin, an experiment was conducted as follows with reference to Fujiwara et al. (Non-patent Document 18) and Fujiwara et al.

免疫化(immunization)は、6週令の雌Balb/cマウス5匹を購入して、alum hydroxide(Sigma) 13mg/mL溶液1.538mL、卵アルブミン10mgおよびPBS 0.4615mLとよく混合して常温で20分間反応させた混合液体をマウス当たり0.2mL(1mg OVA + 2 mg alum)ずつ腹腔に注射したし同じ量を6日目なる日にまた腹腔に注射してブスティング(boosting)した。マウスを13日目犠牲させて脾臟を摘出したしこれから得られた脾臟細胞(splenocyte)100μl(4×106細胞/mL)と試験対象菌の死菌50μlと卵アルブミン50μl(4 mg/ml)を細胞培養ウェルプレート(cell culture well plate)に加えてDMEM-10培地中で7日間10%CO2培養器で培養した。7日間培養した後、上清浄液を取ってIL-12 ELISAキットでアッセイ(Biosource)を遂行してIL-12の濃度を測定した。 For immunization, 5 6-week-old female Balb / c mice were purchased and mixed well with alum hydroxide (Sigma) 13 mg / mL solution 1.538 mL, ovalbumin 10 mg and PBS 0.4615 mL at room temperature. The mixed liquid reacted for 1 minute was injected into the peritoneal cavity by 0.2 mL (1 mg OVA + 2 mg alum) per mouse, and the same amount was boosted by injection into the abdominal cavity on the sixth day. The mouse was sacrificed on the 13th day and the splenic sputum was removed and the splenocyte obtained from this was 100 μl (4 × 10 6 cells / mL), 50 μl of the killed bacteria of the test organism and 50 μl of egg albumin (4 mg / ml) Was added to a cell culture well plate and cultured in a DMEM-10 medium for 7 days in a 10% CO 2 incubator. After culturing for 7 days, the supernatant was removed and assayed (Biosource) with the IL-12 ELISA kit to measure the concentration of IL-12.

前記試験対象菌の死菌は、次の通り獲得した。   The dead bacteria of the test target bacteria were obtained as follows.

験対象菌は、MRS液体培地(Difco)に接種して37℃で24時間培養したし13,000rpmで1分間遠心分離して得た菌体を生理食塩水に2回洗浄後菌体だけ取った。回収された菌体は動物細胞株接種試験のために原培養液体積と同一体積の滅菌蒸溜水で100℃で10分間加熱したし13,000 rpmで1分間遠心分離をした後回収してDMEM培地に適当量希釈して細胞株培養液体積基準として50μg/mlと5μg/mlの濃度の菌体になるようにした。試験対象菌としてラクトバシルロス・プランタラムCJLP56を使ったし、Lactobaillus rhamnosus GG(KCTC 5033)、Lactobacillus casei (KCTC 3109)、Lactobacillus sakei CJLS118(KCTC 13416)に対しても同じ実験を遂行してその結果を比較した。   The test bacteria were inoculated into MRS liquid medium (Difco), cultured at 37 ° C for 24 hours, and centrifuged at 13,000 rpm for 1 minute. . The collected cells were heated in sterile distilled water of the same volume as the original culture volume for 10 minutes at 100 ° C for 10 minutes and centrifuged at 13,000 rpm for 1 minute, and collected to DMEM medium. An appropriate amount was diluted so as to obtain cells having a concentration of 50 μg / ml and 5 μg / ml as a cell line culture volume reference. Lactobaillus rhamnosus GG (KCTC 5033), Lactobacillus casei (KCTC 3109), and Lactobacillus sakei CJLS118 (KCTC 13416) were also used as the test bacteria. Compared.

前記IL-12 ELISAキットを利用したIL-12アッセイ(assay)は、IL-12 ELISAキットに提供された指示事項により実験を進行したしELISA readerで測定されたO.D.値を測定してキットに提供されたIL-12対照サンプルに対する検量式によりIL-12生成量を換算した。そのようにして、得られた測定結果を図4に示した。   The IL-12 assay (assay) using the IL-12 ELISA kit was performed according to the instructions provided in the IL-12 ELISA kit, and the OD value measured by the ELISA reader was measured and provided to the kit. The IL-12 production amount was converted by a calibration formula for the IL-12 control sample. The measurement results thus obtained are shown in FIG.

図4は、ラクトバシルロス・プランタラムCJLP56菌株を卵アルブミンを投与してTh2反応で偏向したマウスの脾臟細胞に処理した後Th1反応誘導サイトキンのIL-12の濃度を測定した結果を他の乳酸菌と比較して示したグラフである。   FIG. 4 shows the results of measurement of IL-12 concentration of Th1 reaction-induced cytokin after treatment of Lactobacillus plantarum strain CJLP56 with spleen splenocytes of mice that were administered ovalbumin and biased by Th2 reaction. It is the graph shown in comparison with lactic acid bacteria.

図4の結果によれば、ラクトバシルロス・プランタラムCJLP56は、Th1反応誘導サイトキンのIL-12の生成を他の乳酸菌に比べて顕著に促進させることが明らかになった。したがって、本発明にともなうラクトバシルロス・プランタラムCJLP56は、Th2反応で偏向したマウスでTh1反応を効果的に誘導するということを確認することができた。   The results of FIG. 4 revealed that Lactobacillus plantarum CJLP56 significantly promoted the production of IL-12, a Th1 reaction-induced cytokin, compared to other lactic acid bacteria. Therefore, it was confirmed that Lactobacillus plantarum CJLP56 according to the present invention effectively induces Th1 response in mice biased by Th2 response.

実施例6:マウス 脾臟細胞処理後IL-4生成抑制力評価
ラクトバシルロス・プランタラムCJLP56(Lactobacillus plantarum CJLP56)菌株を、卵アルブミンを投与してTh2反応で偏向したマウスの脾臟細胞に処理時Th2反応誘導サイトキンのIL-4の生成抑制効果を確認するために、前記実施例5の方法でIL-12キットの代わりにIL-4キット(Biosource)を使うことだけ違って残り条件は同一に実験を進行した。そのようにして測定した結果を図5に示した。
Example 6: Evaluation of inhibitory ability of IL-4 production after treatment of mice splenomegaly cells In order to confirm the inhibitory effect of reaction-induced cytokin on IL-4 production, the remaining conditions were the same except that the IL-4 kit (Biosource) was used instead of the IL-12 kit in the method of Example 5. The experiment proceeded. The measurement results are shown in FIG.

図5は、ラクトバシルロス・プランタラムCJLP56菌株を卵アルブミンを投与してTh2反応で偏向したマウスの脾臟細胞に処理した後Th2反応誘導サイトキンのIL-4の濃度を測定した結果を他の乳酸菌と比較して示したグラフである。   FIG. 5 shows the results of measurement of IL-4 concentration of Th2 reaction-induced cytokin after treatment of Lactobacillus plantarum strain CJLP56 strain with spleen sputum cells that were administered ovalbumin and biased by Th2 reaction. It is the graph shown in comparison with lactic acid bacteria.

図5に示した結果によれば、ラクトバシルロス・プランタラムCJLP56は、Th2反応誘導サイトキンのIL-4サイトキンを抑制することによってTh2反応で偏向したマウス脾臟細胞でTh2反応に対する抑制効果があるということを確認することができた。   According to the results shown in FIG. 5, Lactobacillus plantarum CJLP56 has an inhibitory effect on Th2 response in mouse splenic sputum cells biased by Th2 response by suppressing IL-4 cytokin of Th2 response-induced cytokin. I was able to confirm that there was.

実施例7:マクロファージおよび樹状細胞でTh1リンパ球分化誘導サイトキンIL-12p40およびIL-18の発現とTh1リンパ球分化抑制サイトキンのIL-10発現確認実験
マクロファージおよび樹状細胞のような抗原提示細胞(antigen presenting cell, APC)は、IL-12およびIL-18を生成してTh0リンパ球をTh1リンパ球で分化を誘導して、他の一方ではIL-10を生成してTh1リンパ球で分化誘導を抑制する。本発明にともなう乳酸菌がマクロファージおよび樹状細胞のIL-12、IL-10、IL-18の生成に及ぼす影響を評価するために次のような実験を遂行した。
Example 7: Expression of Th1 lymphocyte differentiation-inducing cytokin IL-12p40 and IL-18 in macrophages and dendritic cells, and IL-10 expression confirmation experiment of Th1 lymphocyte differentiation-inhibiting cytokin Antigens such as macrophages and dendritic cells The presenting cell (antigen presenting cell, APC) produces IL-12 and IL-18 and induces differentiation of Th0 lymphocytes with Th1 lymphocytes, while the other produces IL-10 and Th1 lymphocytes. Suppresses differentiation induction. In order to evaluate the effect of lactic acid bacteria according to the present invention on the production of IL-12, IL-10, and IL-18 by macrophages and dendritic cells, the following experiment was performed.

試験対象菌をマクロファージ細胞株のRAW264.7に5×107/mL濃度で処理して37℃、10% CO2培養を48時間の間遂行した後培地を取ってELISA方法でIL-12p40およびIL-10の濃度を測定した。また、樹状細胞細胞株のJAWSIIにも同じ方法で試験対象菌を接種および培養した後培地を取ってIL-12p40およびIL-10の生成量を測定した。 The test bacteria were treated with RAW264.7, a macrophage cell line, at a concentration of 5 × 10 7 / mL and 37 ° C., 10% CO 2 culture was performed for 48 hours. The concentration of IL-10 was measured. In addition, the dendritic cell line JAWSII was inoculated and cultured in the same manner, and then the culture medium was taken and the production amounts of IL-12p40 and IL-10 were measured.

前記試験対象菌としてラクトバシルロス・プランタラムCJLP56を使ったし、量の対照群としてリポポルリサカライドを使ったし、Lactobaillus rhamnosus GG(KCTC 5033)、Lactobacillus casei (KCTC 3109)、Lactobacillus sakei CJLS118(KCTC 13416)に対しても同じ実験を遂行してその結果を比較した。   Lactobacillus plantarum CJLP56 was used as the test microorganism, and lipopollisacaride was used as a control group, and Lactobaillus rhamnosus GG (KCTC 5033), Lactobacillus casei (KCTC 3109), Lactobacillus sakei CJLS118 The same experiment was performed on (KCTC 13416) and the results were compared.

前記ELISA方法による濃度測定は、IL-12の濃度を測定できるIL-12p40キット(BD Biosciences、USA)およびIL-10の濃度を測定できるIL-10キット(BD Biosciences、USA)を利用して製造会社の指針により遂行した。そのようにして、測定されたそれぞれの結果を図6および図7に示した。   The concentration measurement by the ELISA method is produced using an IL-12p40 kit (BD Biosciences, USA) capable of measuring the concentration of IL-12 and an IL-10 kit (BD Biosciences, USA) capable of measuring the concentration of IL-10. Performed according to company guidelines. The results thus measured are shown in FIG. 6 and FIG.

図6は、ラクトバシルロス・プランタラムCJLP56菌株をマクロファージ細胞株RAW264.7に処理した後、IL-12およびIL-10の濃度をELISAで測定した結果を他の乳酸菌と比較して示したグラフである。   FIG. 6 is a graph showing the results of measuring the concentration of IL-12 and IL-10 by ELISA after treating the Lactobacillus plantarum CJLP56 strain with the macrophage cell line RAW264.7 and comparing it with other lactic acid bacteria. It is.

図7は、ラクトバシルロス・プランタラムCJLP56菌株を樹状細胞細胞株JAWSIIに処理した後、IL-12およびIL-10の濃度をELISAで測定した結果を他の乳酸菌と比較して示したグラフである。   FIG. 7 is a graph showing the results of measuring the concentration of IL-12 and IL-10 by ELISA after treating the Lactobacillus plantarum CJLP56 strain with the dendritic cell line JAWSII and comparing it with other lactic acid bacteria. It is.

図6および7の結果によれば、ラクトバシルロス・プランタラムCJLP56は、Th1分化誘導サイトキンのIL-12サイトキンを生成させて、Th1分化抑制サイトキンIL-10の生成は、IL-12に比べて顕著に少なく生成させるのを確認できるし、他の乳酸菌に比べてIL-12の生成を顕著に増加させるということが分かる。   According to the results of FIGS. 6 and 7, Lactobacillus plantarum CJLP56 generates Th-12 differentiation-inducing cytokin IL-12 cytokin, and Th1 differentiation-inhibiting cytokin IL-10 is produced by IL-12. It can be confirmed that the production is significantly less than that of lactic acid bacteria, and that the production of IL-12 is significantly increased compared to other lactic acid bacteria.

また、遺伝子水準(level)でのIL-12およびIL-18の生成を確認するために試験対象菌をマクロファージ細胞株のRAW264.7に5×107/mL濃度で処理して、37℃、10% CO2培養を6時間の間遂行した後総RNAを抽出してRT-PCR方法でIL-12とIL-18mRNAの生成量を測定した。樹状細胞細胞株のJAWSIIにも同じ方法で試験対象菌を接種および培養した後RNAを抽出してRT-PCR方法でIL-12とIL-18mRNAの生成量を測定した。   In addition, in order to confirm the production of IL-12 and IL-18 at the gene level, the test bacteria were treated with RAW264.7 of the macrophage cell line at a concentration of 5 × 10 7 / mL at 37 ° C., 10 After performing% CO2 culture for 6 hours, total RNA was extracted, and the production amounts of IL-12 and IL-18 mRNA were measured by RT-PCR method. The dendritic cell line JAWSII was inoculated and cultured by the same method, and RNA was extracted, and the production of IL-12 and IL-18 mRNA was measured by RT-PCR.

そのようにして、測定されたそれぞれの結果を図8および図9に示した。   The results thus measured are shown in FIG. 8 and FIG.

図8は、ラクトバシルロス・プランタラムCJLP56菌株をマクロファージ細胞株RAW264.7に処理した後、IL-12p40およびIL-18 mRNAの発現をRT-PCRで測定した結果を他の乳酸菌と比較して示したグラフである。   FIG. 8 shows the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating the Lactobacillus plantarum CJLP56 strain with the macrophage cell line RAW264.7 and comparing it with other lactic acid bacteria. It is the shown graph.

図9は、ラクトバシルロス・プランタラムCJLP56菌株を樹状細胞細胞株JAWSIIに処理した後、IL-12p40およびIL-18mRNAの発現をRT-PCRで測定した結果を他の乳酸菌と比較して示したグラフである。   FIG. 9 shows the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating the Lactobacillus plantarum CJLP56 strain with the dendritic cell line JAWSII in comparison with other lactic acid bacteria. It is a graph.

図8および9の結果によれば、ラクトバシルロス・プランタラムCJLP56は、Th1分化誘導サイトキンのIL-12およびIL-18の生成を指示するmRNAの生成を促進させるということが分かって、特にIL-12 mRNAの発現促進において他の乳酸菌に比べて顕著に優秀だと確認された。   8 and 9, it can be seen that Lactobacillus plantarum CJLP56 promotes the production of mRNA that directs the production of IL-12 and IL-18 of the Th1 differentiation-inducing cytokin, It was confirmed that IL-12 mRNA expression was significantly better than other lactic acid bacteria.

実施例8:ラクトバシルロス・プランタラムCJLP56(Lactobacillus plantarum CJLP56)を含む生菌剤の製造
前記実施例1で同定されたプロバイオティクス ラクトバシルロス・プランタラムCJLP56(Lactobacillus plantarum CJLP56)を医薬品、食品、飼料、飼料添加剤、または化粧品の原料で適用するために大量生産して、これを凍結乾燥して生菌剤化した。
Example 8: Production of probiotic agent containing Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) Probiotic Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) identified in Example 1 They were mass-produced for application with feeds, feed additives, or cosmetic ingredients, and lyophilized into viable bacteria.

菌の生産のためにMRS液体培地(Difco)で25% NaOH溶液を使ってpHを6.0で調節しながら37℃で約18時間培養をして遠心分離を遂行して菌体を回収した。回収した菌体はデキストリン5%と脱脂牛乳10%を凍結保護剤で使って-40℃で凍結後37℃で乾燥した菌体をミキサーで砕いて分体化した。分体化した生菌は目標にする菌数に合わせて保管をするために適当量の葡萄糖、乳糖、脱脂牛乳などと同じ賦形剤と混合して密封されるアルミニウムパウチに入れて包装した。   For the production of the bacteria, the cells were collected by culturing at 37 ° C. for about 18 hours while adjusting the pH to 6.0 with MRS liquid medium (Difco) using a 25% NaOH solution and collecting the cells. The collected cells were frozen using lyoprotectant with 5% dextrin and 10% nonfat milk and frozen at -40 ° C, then dried at 37 ° C and crushed with a mixer. The separated viable bacteria were packaged in a sealed aluminum pouch mixed with the same excipient as sucrose, lactose, skim milk, etc. in order to store them according to the target number of bacteria.

このように製造された生菌剤は飼料に原料で使われる穀物粉と混合して飼料用生菌剤で活用したり、担体または添加剤などを混合して精剤、カプセルなどの医薬品、食品用生菌剤で活用したり、化粧品に使われる原料に一定量を混合することによって医薬品、食品、飼料、化粧品など多様な分野に当該技術分野で通常の方法により活用することができる。   The live bacteria produced in this way is mixed with grain flour used as a raw material in feed and used in live feed for feed, or mixed with carriers or additives, etc. It can be used in a variety of fields such as pharmaceuticals, foods, feeds, cosmetics and the like by ordinary methods in the technical field by using them in live fungi or by mixing a certain amount with raw materials used in cosmetics.

Claims (9)

ラクトバシルロス・プランタラムCJLP56(Lactobacillus plantarumCJLP56) KCTC 11402BP。   Lactobacillus plantarum CJLP56 (KCTC 11402BP). ラクトバシルロス・プランタラムCJLP56(Lactobacillus plantarumCJLP56) KCTC 11402BPを含む腸疾患の予防または治療用組成物。   Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) The composition for the prevention or treatment of an intestinal disease containing KCTC 11402BP. ラクトバシルロス・プランタラムCJLP56(Lactobacillus plantarumCJLP56) KCTC 11402BPを含む免疫増強用組成物。   Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) The composition for immune enhancement containing KCTC 11402BP. Th2反応過剰によるTh1/Th2不均衡に誘発される免疫疾患の予防または治療用であることを特徴とする請求項3に記載の組成物。   The composition according to claim 3, which is used for prevention or treatment of an immune disease induced by Th1 / Th2 imbalance due to an excessive Th2 reaction. 前記免疫疾患は、アレルギー性疾患、アトピー、癌および自家免疫疾患で構成された群で選択されるのを特徴とする請求項4に記載の組成物。   The composition according to claim 4, wherein the immune disease is selected from the group consisting of allergic diseases, atopy, cancer and autoimmune diseases. 医薬品であることを特徴とする請求項2から5のいずれか1項に記載の組成物。   The composition according to claim 2, wherein the composition is a pharmaceutical product. 食品であることを特徴とする請求項2から5のいずれか1項に記載の組成物。   The composition according to any one of claims 2 to 5, wherein the composition is a food. 飼料または飼料添加剤であることを特徴とする請求項2から5のいずれか1項に記載の組成物。   The composition according to any one of claims 2 to 5, which is a feed or a feed additive. 化粧品であることを特徴とする請求項2から5のいずれか1項に記載の組成物。   The composition according to claim 2, wherein the composition is a cosmetic.
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